Hydrogels formed by non-covalent linkages

ABSTRACT

In one aspect, the present invention provides hydrogels comprising polymer molecules and bridging molecules, wherein substantially all the polymer molecules are cross-linked by hydrogen bonds between polymer molecules and bridging molecules, wherein each bridging molecule is linked to at least two polymer molecules, and wherein there are substantially no covalent linkages between the polymer molecules. In some embodiments, the polymer molecules are poly(vinyl alcohol) (PVA) and the bridging molecules are amino acids. Some embodiments of the invention provide devices comprising hydrogels, and pharmaceutical compositions comprising biologically active molecules within hydrogels. Another aspect provides methods for forming hydrogels of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/339,777, filed Jan. 9, 2003, which claims the benefit of U.S.Provisional Application No. 60/347,522, filed Jan. 10, 2002, under 35U.S.C. § 119.

GOVERNMENT RIGHTS

This invention was made with government support under EEC-9529161awarded by the National Science Foundation Engineering Research Center.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to biocompatible hydrogels.

BACKGROUND OF THE INVENTION

Hydrogels are formed by creating bridges between and within polymerchains through the attachment of small bridging molecules to thefunctional moieties of the polymer backbone, a process known ascross-linking. The structural integrity of conventional hydrogels isbased upon the covalent chemistry used for the cross-linking, whichtypically requires catalysts to facilitate the reactions in a timelyfashion. The presence of catalysts impedes the medical use of hydrogels,especially in surgical applications, because they are potentiallyinjurious to surrounding tissues. Thus, there is a need for hydrogelsthat can be polymerized rapidly without the use of chemicalcross-linking catalysts.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides hydrogels comprisingpolymer molecules and bridging molecules, wherein substantially all thepolymer molecules are cross-linked by hydrogen bonds between polymermolecules and bridging molecules, wherein each bridging molecule islinked to at least two polymer molecules and wherein there aresubstantially no covalent linkages between the polymer molecules. Thepolymer molecules are typically neutral polymer molecules with a highdensity of regularly-spaced hydroxyl groups. Exemplary polymer moleculesinclude poly(vinyl alcohol) (PVA), hydroxyethyl acrylate, polyglycerylacrylate, acrylic co-polymers (e.g., TRISACRYL), and polysaccharides.The bridging molecules are typically capable of forming at least twohydrogen bonds. Exemplary bridging molecules include molecules with atleast one of a carboxylic acid group or an amino group. In someembodiments, the bridging molecules are selected from the groupconsisting of amino acids, succinic acid, and ethylene diamine.

The hydrogels of the invention are useful in any situation in which ahydrogel is useful. For example, the hydrogels of the invention can beused to make implantable medical devices wherein the hydrogel portion(s)of the device includes biologically active molecules (e.g., drugs usefulfor treating a disease in a mammal). In some embodiments of theimplantable medical devices the biologically active molecules are notcovalently linked to the hydrogel. In these embodiments, thebiologically active molecules are released from the hydrogel after thedevice is implanted into a living body. Thus, these embodiments ofimplantable medical devices can be used, for example, as drug deliverydevices that release an amount of a drug that is effective to amelioratethe symptoms of a disease in a living body, such as a mammalian body(e.g., a human body).

In some embodiments, the invention provides devices comprising ahydrogel, wherein the hydrogel comprises polymer molecules and bridgingmolecules, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules. In some embodiments, the devicesof the invention further comprise a device body, wherein the hydrogel isattached to the device body. In some embodiments, the devices aremedical devices.

In some embodiments, the devices of the invention are in the form ofpharmaceutical compositions comprising polymer molecules, bridgingmolecules, and biologically active molecules, wherein substantially allthe polymer molecules are cross-linked by hydrogen bonds between polymermolecules and bridging molecules, wherein each bridging molecule islinked to at least two polymer molecules, and wherein there aresubstantially no covalent linkages between the polymer molecules. Thus,the pharmaceutical compositions are useful for delivering biologicallyactive molecules (e.g., therapeutic agents) to a living body, such as amammalian body (e.g., a human body).

In another aspect, the invention provides methods for forming ahydrogel. The methods comprise combining polymer molecules and bridgingmolecules to form a hydrogel, wherein substantially all the polymermolecules are cross-linked by hydrogen bonds between polymer moleculesand bridging molecules, wherein each bridging molecule is linked to atleast two polymer molecules, and wherein there are substantially nocovalent linkages between the polymer molecules. The polymer moleculesare typically neutral polymer molecules with a high density ofregularly-spaced hydroxyl groups. Exemplary polymer molecules includepoly(vinyl alcohol) (PVA), hydroxyethyl acrylate, polyglyceryl acrylate,acrylic co-polymers (e.g., TRISACRYL), and polysaccharides. The bridgingmolecules are typically capable of forming at least two hydrogen bonds.Exemplary bridging molecules include molecules with at least one of acarboxylic acid group or an amino group. In some embodiments, thebridging molecules are selected from the group consisting of aminoacids, succinic acid, and ethylene diamine. The methods of this aspectof the invention are useful for forming the hydrogels of the presentinvention.

Some embodiments provide methods for forming a hydrogel at a site ofapplication are provided. The methods for forming a hydrogel at a siteof application comprise combining polymer molecules and bridgingmolecules at the site of application to form a hydrogel, whereinsubstantially all the polymer molecules are cross-linked by hydrogenbonds between polymer molecules and bridging molecules, wherein eachbridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules. The methods of this aspect of the invention are useful, forexample, for forming hydrogels at the site of a wound in a living body,such as a mammalian body (e.g., a human body). The hydrogel may fill in,or repair, a missing or damaged portion of tissue, and/or may deliverbiologically active molecules (e.g., therapeutic agents) to a damagedportion of a living body, thereby promoting wound healing. In someembodiments, the methods of this aspect of the invention may be used toapply stem cells, included within a hydrogel of the invention, to adamaged portion of a living body. The stem cells thereafter divide anddifferentiate to form cells and tissue that repair the damaged portionof the living body.

Further embodiments provide methods of making a pharmaceuticalcomposition comprising a hydrogel and biologically active molecules. Themethods of making a pharmaceutical composition comprise combiningbiologically active molecules with polymer molecules and bridgingmolecules to form a hydrogel comprising the biologically activemolecules, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules. The pharmaceutical compositionsof the invention are useful, for example, for delivering therapeuticagents to a living body in need thereof (e.g., for delivering an amountof a drug effective to treat a disease afflicting a living body, such asa mammalian body).

In another aspect, the invention provides methods for administeringbiologically active molecules to a subject, comprising administering tothe subject biologically active molecules in a hydrogel of theinvention. Some embodiments provide methods for injecting biologicallyactive molecules, comprising the steps of: (a) applying a layer ofhydrogel to a site of injection, wherein substantially all the polymermolecules are cross-linked by hydrogen bonds between polymer moleculesand bridging molecules, wherein each bridging molecule is linked to atleast two polymer molecules, and wherein there are substantially nocovalent linkages between the polymer molecules; and (b) injectingbiologically active molecules through the hydrogel layer. An advantageof the methods of this aspect of the invention is that the injectedmolecules are prevented, by the layer of hydrogel, from escaping fromthe site of injection after the needle, or other instrument used toinject them, is removed.

A further aspect provides kits for forming the hydrogels of theinvention. The kits comprise polymer molecules, bridging molecules, andinstructions for forming a hydrogel. The instructions provide protocolsfor combining the polymer molecules and bridging molecules to form ahydrogel wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules. Thus, for example, the kits ofthe invention can be used to form the hydrogels of the invention. Forexample, in some embodiments, the kits can be used to form a hydrogel inthe methods of the invention for forming a hydrogel at a site ofapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a schematic representation of the hydrogen bonding betweenpoly(vinyl alcohol) and both the amino group of glycine and the carboxylgroup of glycine in a representative hydrogel of the invention (R═CH₂).

FIG. 2 shows a representative shaped hydrogel article of the invention,a contact lens.

FIG. 3 shows a graph representing the extent of complement activation byrepresentative hydrogels of the invention, measured as the amount ofSC5b-9 present as described in EXAMPLE 5.

FIG. 4 shows a perspective view of a representative medical device ofthe invention with a portion of the hydrogel layer removed to expose theunderlying device body.

FIG. 5 shows a transverse cross-section of the medical device of FIG. 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one aspect, the present invention provides hydrogels comprisingpolymer molecules and bridging molecules, wherein substantially all thepolymer molecules are cross-linked by hydrogen bonds between polymermolecules and bridging molecules, wherein each bridging molecule islinked to at least two polymer molecules and wherein there aresubstantially no covalent linkages between the polymer molecules.

It is a feature of the hydrogels of the invention that they do not relyon covalent bonds between polymer molecules to provide structuralstability. The structural stability of the hydrogels of the invention isprovided, for the most part, by the hydrogen bonds formed betweenbridging molecules and polymer molecules whereby the polymer moleculesare cross-linked. Thus, each bridging molecule forms hydrogen bonds withat least two polymer molecules, thereby cross-linking the polymermolecules. Consequently, the hydrogels of the invention typically do notinclude any catalyst that is used in art-recognized processes for makinghydrogels by covalently cross-linking polymer molecules. Many of thesecatalysts are toxic or otherwise deleterious to living cells or tissue.Thus, the hydrogels of the invention are typically more biocompatiblethan prior art hydrogels that include residual amounts of catalysts usedto form covalent bonds between polymer molecules.

The polymer molecules are typically neutral molecules having a highdensity of regularly-spaced hydroxyl groups. Thus, the polymer moleculecan be any polymer, natural or synthetic, in which there areregularly-spaced hydroxyl moieties that can form hydrogen bonds with alow molecular weight bridging molecule, for example as shown in FIG. 1.Suitable polymers include poly(vinyl alcohol) (PVA), hydroxyethylacrylate, polyglyceryl acrylate, polysaccharides, and acrylicco-polymers such poly(N-tris[hydroxymethyl]methyl) acrylamide (e.g.,TRISACRYL, Sigma/Aldrich).

In the hydrogels of the invention, the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, as shown, for example, in FIG. 1. Each bridging molecule islinked to at least two polymer molecules. Thus, the physical integrityof the hydrogel is dependent on the hydrogen bonds formed between thepolymer molecules and the bridging molecules. There are substantially nocovalent linkages between the polymer molecules, although some covalentlinkages between the polymer molecules may form spontaneously at a lowrate. Thus, typically no more than about 1% (more typically no more thanabout 0.1%) of the polymer molecules are linked by one or more covalentbonds.

The bridging molecule can be any low molecular weight molecule (m.w.<1000) that can form at least two hydrogen bonds with the polymermolecules. As used herein, the term “low molecular weight molecule”refers to a molecule with a molecular weight that is less than 1000. Atleast two hydrogen bonds are necessary to connect two different polymermolecules in order to obtain cross-linking. The bridging moleculetypically includes one or more carboxylic acid groups and/or one or moreamino groups. Thus, many low molecular weight molecules are suitable foruse in the invention, such as naturally-occurring or synthetic aminoacids. Representative examples of useful amino acids include: threonine,serine, tyrosine, phenylalanine, proline, histidine, glycine, lysine,alanine, arginine, cysteine, tryptophan, valine, glutamine, and asparticacid, as described in EXAMPLE 3. Suitable bridging molecules may alsoinclude other zwitterionic molecules, or any molecule that comprises atleast one of a carboxylic acid group or an amino group, such as succinicacid or ethylene diamine, as described in EXAMPLE 4.

In some embodiments, the bridging molecule is an amino acid or a mixtureof amino acids, as described in EXAMPLES 1-3. Both L- and D-isomers ofamino acids can be included in the hydrogels of the invention, as shownin EXAMPLE 3. Many useful functional groups can be inserted into thehydrogel by virtue of the chemistry of the side chain of amino acids.This offers the means to covalently immobilize proteins, drugs and otherbiologically active molecules that interact with cells migrating intothe gel from surrounding tissue. The functional groups on the amino acidside chains can be chemically coupled by a variety of commonchemistries, including, but not limited to, carbodiimides, aryl azides,and succinimidyl esters or carbonyldiimidazole derivatization.Hydrolyzable linkage chemistry or reversible modifications could also beemployed for local or systemic delivery of biologically activemolecules.

Covalent linkages can be formed between any suitable chemical groupspresent in amino acid molecules within the hydrogels of the invention.For example, amino acids that are easily derivatizable at their sidechains include aspartic acid, glutamic acid, lysine, arginine, cysteine,histidine, tyrosine, methionine and tryptophan. These nine amino acidscontain the following eight principal functional groups that can becovalently linked: primary amines (NH₂), carboxylate (COOH), sulfhydrylsor disulfides (SH or S-S), thioethers (found in methionine), imidazolyls(found in histidine), guanidinyl groups (found in arginine), phenolic(found in tyrosine), and indolyl (found in tryptophan). These functionalgroups can be covalently linked using any suitable chemical reaction,such as the methods disclosed in Hermanson, Bioconjugate Techniques,Academic Press (1996), which publication is incorporated herein byreference. Other amino acids, such as asparagine, glutamine, threonine,and serine may also be covalently link through amide and/or hydroxylfunctional groups, as described in Hermanson, Bioconjugate Techniques,Academic Press (1996).

Desired properties of the hydrogels of the invention can be obtained,for example, by varying stoichiometric ratio of the polymer and thebridging molecule, as described in EXAMPLE 2. In general, hydrogelscomprising higher concentrations of bridging molecules are firmer thanhydrogels comprising lower concentrations of bridging molecules. Thus,hydrogels comprising PVA and amino acids generally increase in firmnesswith increasing amino acid concentration, as shown in EXAMPLE 1. As usedherein, the term “firm hydrogel” refers to a hydrogel that retains itsshape. Typically, a firm hydrogel may be squeezed or pinched, however,it will bounce back to its original form when released. The term “softhydrogel” refers to a hydrogel that is a viscous liquid or has atoothpaste-like consistency. A soft hydrogel typically adapts the formof the container it is in. For example, a hydrogel formed with PVA andglycine at a ratio of 0.304 (PVA:Gly, w/w) has a soft “toothpaste”consistency, whereas a hydrogel formed with PVA and glycine at a ratioof 0.201 (PVA:Gly, w/w) is firm and rubbery. On the other hand, ahydrogel formed with PVA and tryptophan at a ratio of 12.5 (PVA:Trp,w/w) is firm and rubbery. Thus, the ratio of polymer to bridgingmolecule used to make a firm or a soft hydrogel varies according to thepolymer molecule and bridging molecules used. Suitable ratios of polymermolecules to bridging molecules to provide hydrogels of a desiredconsistency can readily be determined empirically, as described inEXAMPLES 1-3.

In some embodiments, the invention provides devices comprising ahydrogel, wherein the hydrogel comprises polymer molecules and bridgingmolecules, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules.

The devices comprising a hydrogel may include, but are not limited to,pill and gel formulations, films at material surfaces, ointments fortopical use, and shapes that conform to the contours of the surface(e.g., a body part) that they are applied to (e.g., shapes that arewound-filling or defect-filling). FIG. 2 shows a representative deviceof the invention, in the form of a contact lens 10 having an innersurface 12 and an outer surface 14.

Some hydrogels of the invention provide excellent integration withsurrounding tissue upon implantation in vivo, as described in EXAMPLE 5.These hydrogels are biocompatible and elicit no or minimal unfavorableresponses, such as inflammation, foreign body encapsulation, orcomplement system activation (FIG. 3, EXAMPLE 5). The hydrogels of theinvention can be manufactured from raw materials that have been acceptedas safe for medical use by the United States Pharmacopia (USP).Accordingly, they may be used for a variety of medical applicationsincluding, but not limited to, surgical adhesives, sealants, andbarriers, coatings, lubricants, adhesion-preventing formulations,transducers for ultrasound imaging from both outside and within the bodyduring surgery, hemostasis control materials, and medical devicecoatings. An exemplary use of a hydrogel of the invention to create abarrier to prevent the extrusion of biologically active molecules fromthe site of injection is provided in EXAMPLE 7.

Some embodiments of the devices of the invention provide implantabledelivery systems for drugs, biologicals and other biologically activemolecules. An exemplary pharmaceutical composition for delivery ofbiologically active molecules to a subject is described in EXAMPLE 6.The pharmaceutical compositions of the invention can be used toimmobilize covalently a variety of proteins, drugs, and otherbiologically active molecules for presentation to the surrounding cellsthat invade the gel. Controlled delivery of drugs and other biologicallyactive molecules, such as DNA, RNA, or proteins can be achieved by usinghydrolyzable linkages or reversible modifications.

In some embodiments, the invention provides devices in the form ofpharmaceutical compositions, comprising biologically active molecules ina hydrogel of the invention. Administration of the pharmaceuticalcompositions of the invention is accomplished by any effective route,e.g., orally or parenterally. Methods of parenteral delivery includetopical, intra-arterial, subcutaneous, intramedullary, intravenous, orintranasal administration. In addition to one or more biologicallyactive molecules, the pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients and othercompounds that facilitate administration of the biologically activemolecules to a subject. Further details on techniques for formulationand administration may be found in the latest edition of “Remington'sPharmaceutical Sciences” (Maack Publishing Co, Easton Pa.).

In some embodiments, the devices of the invention further comprise adevice body, wherein the hydrogel is attached to the device body. Someembodiments provide medical devices comprising a device body and ahydrogel of the invention attached to the device body. The hydrogel maybe immobilized onto (or within) a surface of an implantable orattachable medical device body. The modified surface will typically bein contact with living tissue after implantation into an animal body. Asused herein, “implantable or attachable medical device” refers to anydevice that is implanted into, or attached to, tissue of an animal body,during the normal operation of the device (e.g., implantable drugdelivery devices). Such implantable or attachable medical device bodiescan be made from, for example, nitrocellulose, diazocellulose, glass,polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran,Sepharose, agar, starch, and nylon. Linkage of the hydrogel to a devicebody can be accomplished by any technique that does not destroy thedesired properties of the hydrogel. For example, hydrogels comprisingamino acids may be attached to the device body through functional groupsof the amino acids. A surface of an implantable or attachable medicaldevice body can be modified to include functional groups (e.g.,carboxyl, amide, amino, ether, hydroxyl, cyano, nitrido, sulfanamido,acetylinic, epoxide, silanic, anhydric, succinimic, azido) for hydrogelimmobilization thereto. Coupling chemistries include, but are notlimited to, the formation of esters, ethers, amides, azido andsulfanamido derivatives, cyanate and other linkages to the functionalgroups available on the hydrogels.

In some embodiments, a surface of a device body that does not possessuseful reactive groups can be treated with radio-frequency dischargeplasma (RFGD) etching to generate reactive groups (e.g., treatment withoxygen plasma to introduce oxygen-containing groups; treatment withpropyl amino plasma to introduce amine groups). When an RFGD glowdischarge plasma is created using an organic vapor, deposition of apolymeric overlayer occurs on the exposed surface. RFGD plasma depositedfilms offer several unique advantages. They are smooth, conformal, anduniform. Film thickness is easily controlled and ultrathin films(10-1000 Angstroms) are readily achieved, allowing for surfacemodification of a material without alteration to its bulk properties.Moreover, plasma films are highly-crosslinked and pin-hole free, andtherefore chemically stable and mechanically durable. RFGD plasmadeposition of organic thin films has been used in microelectronicfabrication, adhesion promotion, corrosion protection, permeationcontrol, as well as biomaterials (see, e.g., U.S. Pat. No. 6,131,580).

Some medical devices of the invention are adapted to be implanted intothe soft tissue of an animal, such as a mammal, including a human,during the normal operation of the medical device. Implantable medicaldevices of the invention may be completely implanted into the softtissue of an animal body (i.e., the entire device is implanted withinthe body), or the device may be partially implanted into an animal body(i.e., only part of the device is implanted within an animal body, theremainder of the device being located outside of the animal body).Representative examples of completely implantable medical devicesinclude, but are not limited to: cardiovascular devices (such asvascular grafts and stents), artificial blood vessels, artificial bonejoints, such as hip joints, and scaffolds that support tissue growth (insuch anatomical structures as nerves, pancreas, eye and muscle).Representative examples of partially implantable medical devicesinclude: biosensors (such as those used to monitor the level of drugswithin a living body, or the level of blood glucose in a diabeticpatient) and percutaneous devices (such as catheters) that penetrate theskin and link a living body to a medical device, such as a kidneydialysis machine.

Some medical devices of the invention are adapted to be affixed to softtissue of an animal, such as a mammal, including a human, during thenormal operation of the medical device. These medical devices aretypically affixed to the skin of an animal body. Examples of medicaldevices that are adapted to be affixed to soft tissue of an animalinclude skin substitutes, and wound or burn treatment devices (such assurgical bandages and transdermal patches).

FIG. 4 shows a representative medical device 10 of the presentinvention, in the form of an implantable drug delivery device, whichincludes a device body 12 to which is attached a hydrogel layer 14. Inthe embodiment shown in FIG. 4, hydrogel layer 14 has been partiallyremoved to show device body 12 beneath. Device body 12 is indicated byhatching. As shown in the cross-sectional view of medical device 10 inFIG. 5, hydrogel layer 14 includes an internal surface 18, attached todevice body 12, and an external surface 20.

Due to the biocompatibility of the hydrogels of the invention used inthe construction of medical device 10, the presence of the hydrogel onthe device body of a medical device will reduce or eliminate the foreignbody response to the device body after implantation into, or attachmentto, tissue of an animal body.

In some embodiments, the medical devices of the invention furthercomprise biologically active molecules within the hydrogel attached tothe device body to provide for the controlled delivery of drugs andother biologically active molecules, such as DNA, RNA, or proteins. Thebiologically active molecules may be attached, covalently ornon-covalently, to the bridging molecules (e.g., amino acids) and/or tothe polymer molecules in the hydrogel. Examples of functional groupsuseful for covalently attaching biologically active molecules to aminoacids present within the hydrogel include: primary amines (NH₂),carboxylate (COOH), sulfhydryls or disulfides (SH or S-S), thioethers,imidazolyls, guanidinyl groups, phenolic, and indolyl.

Any reactive functional group present on polymer molecules within thehydrogel can be used to covalently attach biologically active moleculesto the hydrogel. The following publications, incorporated herein byreference, describe examples of technologies that are useful forattaching biologically active molecules to polymer molecules, such asthe polymers present in the hydrogel of the present invention: Nuttelmanet al. (2001) J. Biomed. Mater. Res. 57:217-223; Rowley et al. (1999)Biomaterials 20:45-53; Hubbel (1995) Biotechnology 13:565-76; Massia &Hubbell (1990) Anal. Biochem 187:292-301; Drumheller et al. (1994) Anal.Biochem. 222:380-8; Kobayashi & Ikada (1991) Curr. Eye Res. 10:899-908;Lin et al. (1992) J. Biomaterial Sci. Polym. Ed. 3:217-227; andBellamkonda et al. (1995) J. Biomed. Mater. Res. 29:663-71.

The biologically active molecules may also be introduced into thehydrogel by forming the hydrogel in the presence of the biologicallyactive molecules, by allowing the biologically active molecules todiffuse into a hydrogel, or by otherwise introducing the biologicallyactive molecules into the hydrogel (e.g., by injection, as described inEXAMPLE 6).

The biologically active molecules can be attached to every part of thedevice, or to only a portion of the device. For example, in someembodiments, that are adapted to be implanted into an animal,biologically active molecules that act to decrease the foreign bodyreaction (e.g., anti-inflammatory agents, and immunomodulatory agents)are attached only to the surface(s) of the device that is/are in contactwith living tissue in the animal body. The biologically active moleculesserve to decrease the foreign body reaction of the living body againstthe implanted structure.

In another aspect, the invention provides methods for forming ahydrogel. The methods comprise combining polymer molecules and bridgingmolecules to form a hydrogel, wherein substantially all the polymermolecules are cross-linked by hydrogen bonds between polymer moleculesand bridging molecules, wherein each bridging molecule is linked to atleast two polymer molecules, and wherein there are substantially nocovalent linkages between the polymer molecules.

The polymer molecules are typically neutral molecules having a highdensity of regularly-spaced hydroxyl groups. Thus, the polymer moleculecan be any polymer, natural or synthetic, in which there areregularly-spaced hydroxyl moieties that can form hydrogen bonds with alow molecular weight bridging molecule, as shown in FIG. 1. Suitablepolymers include poly(vinyl alcohol) (PVA), hydroxyethyl acrylate,polyglyceryl acrylate, polysaccharides, and acrylic co-polymers suchpoly(N-tris[hydroxymethyl]methyl) acrylamide (e.g., TRISACRYL,Sigma/Aldrich).

The polymer molecules are cross-linked by hydrogen bonds between polymermolecules and bridging molecules, as shown, for example, in FIG. 1. Eachbridging molecule is linked to at least two polymer molecules, and thereare substantially no covalent linkages between the polymer molecules.Typically, no more than about 1% of the polymer molecules are covalentlylinked (more typically no more than about 0.1% of the polymer moleculesare covalently linked).

The bridging molecule can be any low molecular weight molecule that canform at least two hydrogen bonds with the polymer molecules. Thebridging molecule typically includes one or more carboxylic acid groupsand/or one or more amino groups. Thus, many low molecular weightmolecules are suitable for use in the invention, such asnaturally-occurring or synthetic amino acids. Representative examples ofuseful amino acids include: threonine, serine, tyrosine, phenylalanine,proline, histidine, glycine, lysine, alanine, arginine, cysteine,tryptophan, valine, glutamine, and aspartic acid, as described inEXAMPLE 3. Suitable bridging molecules also include other zwitterionicmolecules, or any molecule that comprises at least one of a carboxylicacid group or an amino group, such as succinic acid or ethylene diamine,as described in EXAMPLE 2.

In some embodiments, the bridging molecule is an amino acid or a mixtureof amino acids, as described in EXAMPLES 1-3. Virtually any naturallyoccurring or synthetic amino acid can be employed to make the hydrogelsof the invention. In some embodiments, hydrogels are formed by combiningpoly(vinyl alcohol) (PVA) solutions with glycine, bicine(N,N-bis(2-hydroxyethyl)glycine), glutamine, cysteine, arginine, lysine,histidine, and trans-4-hydroxyl proline, serine, methionine, ortryptophan. Mixtures of amino acids can also be used, such as glycineand lysine in combination with PVA, or glycine and arginine incombination with PVA, as described in EXAMPLE 4.

In some embodiments, hydrogels are formed by combining PVA and aminoacids at a ratio of 8-200 mg of amino acids to 1 ml of 8-10% PVA. PVA isobtained from poly(vinyl acetate) through acid hydrolysis converting theacetate groups into alcohol functions. In some embodiments, the PVA ismore than 80% hydrolyzed. In some embodiments, the PVA is more than 90%hydrolyzed. In some embodiments, the PVA is more than 95% hydrolyzed.Hydrogels are typically formed within 24 hours at room temperature, andmay take up to a week to reach equilibrium. The basis for the hydrogelformation in the absence of catalyzed cross-linking lies, at least inpart, in the ability of the amino acids to form hydrogen bonds with theOH groups in the PVA.

Both L- and D-isomers of amino acids can be included in the hydrogels ofthe invention, as shown in EXAMPLE 3. Many useful functional groups canbe inserted into the hydrogels by virtue of the chemistry of the sidechain of amino acids, as described above. This offers the means tocovalently immobilize proteins, drugs and other biologically activemolecules.

The invention provides methods for forming hydrogels having desiredphysical or chemical properties. As described above, useful functionalgroups can be inserted into the hydrogel by virtue of the chemistry ofbridging molecules, such as the side chains of amino acids. Moreover,the desired physical properties of the hydrogels, such as gelling speed,firmness, and solubility, can be precisely adjusted by controlling thepH, the temperature, and the stoichiometric ratio of the polymer and thebridging molecule, as described in EXAMPLES 1 and 2. The affinity of thehydrogel for water, plasma, blood, and cellular components can also bevaried by changing the nature and the ratio of the component moleculesof the hydrogel. Thus, the conditions suitable for forming a hydrogelwith the desired properties depend on the choice of bridging molecule,the ratio of polymer to bridging molecule, the pH, and the desiredphysical properties of the hydrogel.

The interaction between the polymer molecule and the bridging moleculeoccurs when the bridging molecule is deprotonated. Therefore, a suitablepH range for forming the hydrogel of the invention is the pH range atwhich most or all of the bridging molecules are deprotonated, asdescribed in EXAMPLE 2. Accordingly, the choice of pH will depend on thenature of the bridging molecule(s) used for forming the hydrogel.

Hydrogels comprising both PVA and one or more amino acids are stable forseveral months in water, in contrast to hydrogels comprising PVA alone,which degrade after a few days. In general, hydrogels comprising higherconcentrations of bridging molecules are firmer than hydrogelscomprising lower concentrations of bridging molecules, as described inEXAMPLE 2. For example, a hydrogel formed with PVA and glycine at aratio of 0.304 (PVA:Gly, w/w) has a soft “toothpaste” consistency,whereas a hydrogel formed with PVA and glycine at a ratio of 0.201(PVA:Gly, w/w) is firm and rubbery. On the other hand, a hydrogel formedwith PVA and tryptophan at a ratio of 12.5 (PVA:Trp, w/w) is firm andrubbery. Thus, the ratio of polymer to bridging molecule used to make afirm or a soft hydrogel varies according to the polymer molecule andbridging molecules used. Suitable ratios of polymer molecules tobridging molecules to provide hydrogels of a desired consistency canreadily be determined empirically, as described in EXAMPLES 1-3.

In some embodiments, the invention provides methods for forming ahydrogel at a site of application. The methods for forming a hydrogel ata site of application comprise combining polymer molecules and bridgingmolecules at the site of application to form a hydrogel, whereinsubstantially all the polymer molecules are cross-linked by hydrogenbonds between polymer molecules and bridging molecules, wherein eachbridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules. The site of application refers to the place at or near wherethe hydrogel is to be applied. For example, the site of application maybe in or on a subject, such as a human subject. According to themethods, instead of applying a previously formed hydrogel to the site,the hydrogel is formed at that site (in situ). The components of thehydrogel may be applied separately to the site of application andallowed to mix and gel in situ. Alternatively, the components may firstbe mixed and then applied to the site of application and allowed to gelin situ, as described in EXAMPLE 7. A hydrogel may be formed in situ,for example, by using a dual syringe or tube delivery assemblycontaining the components of the hydrogel in separate chambers. Anexemplary embodiment of a dual syringe delivery assembly contains twosyringes connected by a needle. The first syringe contains a polymermolecule solution and the second syringe contains a bridging moleculesolution. The two solutions are combined by injecting, for example, thepolymer molecule solution in the first syringe into the second syringe,then injecting the combined polymer molecule and bridging molecule inthe second syringe into the first syringe. The injections from onesyringe into the other syringe are continued until both solutions aremixed, after which the empty syringe is discarded, and the mixedsolution is extruded through the needle.

Further embodiments provide methods of making a pharmaceuticalcomposition comprising a hydrogel and biologically active molecules. Themethods of making a pharmaceutical composition comprise combiningbiologically active molecules with polymer molecules and bridgingmolecules to form a hydrogel comprising the biologically activemolecules, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules. The methods comprise mixing thebiologically active molecules with one or more components of thehydrogel before the hydrogel is formed. The methods may also compriseadding biologically active molecules to an already formed hydrogel. Forexample, the biologically active molecules may be absorbed into thehydrogel by osmosis, or they may be injected, as described, for example,in EXAMPLE 6.

Another aspect provides methods for administering the hydrogels of theinvention. In some embodiments, the hydrogels comprise biologicallyactive molecules. An exemplary method for administering a hydrogelcomprising biologically active molecules is described in EXAMPLE 6.

Some embodiments provide methods for injecting biologically activemolecules, comprising the steps of: (a) applying a layer of hydrogel toa site of injection, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules; and (b) injecting biologicallyactive molecules through the hydrogel layer. An exemplary method forinjecting biologically active molecules according to the invention isdescribed in EXAMPLE 7.

The methods for administering a hydrogel include any route ofadministration, including oral and parenteral, such as topical,transdermal, nasal, vaginal, rectal, or sublingual routes ofadministration, intramuscular, sub-cutaneous, intravenous, andintraperitoneal. The hydrogels can be formulated in dosage formsappropriate for each route of administration.

Another aspect of the invention provides kits for forming the hydrogelsof the invention. The kits comprise polymer molecules, bridgingmolecules, and instructions for forming one or more hydrogels of theinvention. The polymer molecules and bridging molecules in the kit maybe provided as a dehydrated or lyophilized mixture that can bereconstituted to form a hydrogel. The polymer molecules and bridgingmolecules may also be provided as solutions that may be combined to forma hydrogel. The instructions provided with the kit provide protocols forforming a hydrogel with the polymer molecules and bridging moleculesprovided in the kit. For example, there may be a chart listingapplications types and appropriate ratios of polymer molecules andbridging molecules for each application. Some embodiments of the kitfurther provide a container in which the hydrogel may be formed. Thecontainer may be a mold, one or more syringes, or any other kind ofcontainer.

EXAMPLE 1

This Example describes the formation of representative hydrogels of theinvention by combining poly(vinyl alcohol) and glycine or cysteine.

An 8% (w/v) solution of poly(vinyl alcohol) (PVA) (MW 86,000-140,000;Aldrich Chemicals, 99% hydrolyzed) had an approximate viscosity of 130cps (Centipoise) at 23° C. as measured with a Brookfield Model DV-IIdigital viscometer employing a spindle number 31, and small sampleadaptor, with a shear rate of 10 rpm. Addition of the amino acidglycine, dissolved in 2 ml water, to 10 ml of the 8% PVA solution causedgelation associated with a subsequent increase in viscosity inproportion to the amount of glycine added, as shown in Table 1. Theviscosity was measured after 5 minutes of stirring and stabilization ofthe viscometer. TABLE 1 Viscosity Changes During PVA/Gly HydrogelFormation Amount of Glycine Added (g) Viscosity (cP) 0 130 0.25 153 0.4159 0.5 144 0.75 192 0.9 186

In a second experiment, the PVA was re-precipitated with acetone tofurther purify it. Different ratios of an 8% solution of re-purified PVAand a 30% solution of glycine were combined in a total of 10 ml at roomtemperature. Viscosity measurements were taken 100 seconds after the twosolutions were combined using a digital viscometer (Brookfield ModelDV-II), spindle # 31 with a shear rate of 50 rpm, and the small adaptor.The viscosity measurements are provided in Table 2. TABLE 2 ViscosityChanges During Hydrogel Formation with Re-Purified PVA/Gly Ratio ofGly:PVA (w/w) Viscosity (cP) 1  86 2 124 3 167 4 208 5  652**shear rate had to be adjusted to 20 rpm

These experiments show that the viscosity of the hydrogel can easily becontrolled by varying the ratio of PVA to glycine.

EXAMPLE 2

This Example describes that bridging molecules containing either acarboxylic acid or an amino group are capable of complexing with PVA tocause formation of representative hydrogels of the invention.

Two model compounds, succinic acid and ethylene diamine, were chosen,each of which only contained either two carboxylic acid groups (succinicacid) or two amino groups (ethylene diamine).

Succinic acid, ethylene diamine, and glycine were each dissolved inwater at a typical ratio of 100 mg in 1 ml water. The pH of the compoundsolution (natural pH) was measured, and, if adjusted to a different pH(typically pH 4, 7, and 10), 5M NaOH_((aq)) or 5M HCl_((aq)) was addeduntil the desired pH was reached.

Aliquots of 100 microliter of each solution were added to PVA to reachfinal concentrations of either ˜0.5 mmol (low concentration) or ˜1 mmol(high concentration) per 1 ml 8% PVA solution (1.8 mmol of available OHgroups in PVA). PVA gels containing succinic acid and ethylene diaminewere compared to PVA/glycine (PVA/Gly) gels prepared under similarconditions (see Table 3).

Control: As a control, 10 to 100 microliters of either 5M HCl or 5M NaOHwere added to 1 ml of 8% PVA. The amounts added to PVA were similar tothe volume of acid or base added to PVA. No gel formation was observedwith acidic water. The basic solution causes PVA to thicken slightly. Alittle additional gelling was observed within 24 hours but it was notsubstantial.

Glycine: Glycine's natural pH is 6.78. At pH 4, little gel formation wasobserved initially; however, additional gel formation was observedwithin the first 24 hours.

At pH 7, substantial gelling was observed upon addition of glycine. Moregelling was observed over time. At higher concentrations of glycine,immediate gel formation was observed with phase separation. Theprecipitated PVA was very soft and pliable, but was firmer if a higherconcentration of glycine is used. The gel did not dissolve if it wasallowed to cure for 1 hour in the solution it was made in before placingit in water. At pH 7, 2.6 mmol of glycine (2.6 mmol of each COO⁻ andNH₂) is necessary to cause precipitation of 1 ml 8% PVA (1.8 mmol ofavailable OH groups).

At pH 9.9, much gelling was observed upon initial addition. Considerableadditional gelling was observed within 24 hours.

Succinic Acid: At pH 2.1, very slight gelling was observed after initialaddition, however, the gelling dispersed easily with mixing. Noadditional gelling was observed with time.

At pH 7.0, there was some initial gelling when small amounts were added.A little more gelling was observed over time. At higher concentration ofsuccinic acid, PVA precipitates out immediately upon addition ofsuccinic acid, and the gel formed is typically in the form of a soft andwhite lump. This gel is similar to that obtained using glycine, however,the gel forms faster and is harder at end. The gel did not dissolve inwater or in 1M HCl.

At pH 9.7, there was some initial gelling when small amounts are added.Additional gelling was observed over time. At higher concentration ofsuccinic acid, PVA precipitates out immediately upon addition ofsuccinic acid, and the gel formed is typically in the form of a soft andwhite lump. This gel is similar to that obtained using glycine, however,the gel forms faster and is harder at end. The gel did not dissolve inwater or in 1M HCl. It took slightly less succinic acid than at pH 7 toinitiate gel formation.

At pH 7, 0.85 mmol of succinic acid is necessary (1.7 mmol of COO⁻) tocause precipitation of 1 ml 8% PVA (1.8 mmol of available OH groups). AtpH 10, 0.73 mmol of succinic acid is necessary (1.5 mmol of COO⁻) tocause precipitation of 1 ml 8% PVA (1.8 mmol of available OH groups).

Ethylene Diamine: At pH 4.4, no immediate gelling was observed, andthere was no gelling over time. At pH 7.2, there was little immediategelling, and no additional gelling with time. At pH. 10.0 and at pH12.7, there was immediate gelling with lump upon mixing formation; andadditional gelling occurred during the first 24 hours.

These results show that compounds only containing carboxylic groups willgel PVA. They can be more effective than amino acids. The carboxylicacid interacts with PVA when it is deprotonated and exists as COO⁻. Theresults also show that compounds containing only amino groups result ingelling of PVA in a similar way as basic amino acids, such as lysine andarginine. The amino group interacts with PVA when it is in itsdeprotonated state, as NH₂. TABLE 3 Gel Formation with Glycine, SuccinicAcid and Ethylene Diamine. pH 2.1 pH 4 pH 7 pH 10 pH 13.1 Glycine n.t. −+++ ++ n.t. Succinic Acid − − +++ +++ n.t. Ethylene Diamine n.t. − − +++++n.t. = Compound was not tested at that pH;+ = Gel formation, the more + the more gelling;− = No gel formation was observed.

EXAMPLE 3

This Example describes studies showing that both D-amino acids andL-amino acids can be used to form hydrogels of the invention.

In these experiments, the ability of D-amino acids to gel PVA wascompared directly to the gelling observed using L-amino acids. Theconcentrations of amino acids used were the same for both the L- andD-forms. All D- and L-amino acids were obtained from Aldrich. Typically,100 mg of amino acid was dissolved in the minimum amount of water or 1MNaHCO₃ (200-600 microliter) necessary to dissolve it. Even with 600microliter of solvent, some amino acids did not dissolve upon heating;in these cases, the supernatant of the solution was used. For each aminoacid/PVA combination, 1 ml of 8% PVA was used (80 mg PVA) and the amountof amino acid was varied. 100 mg of each of amino acids was added unlesslisted below, with the following exceptions: L-Gln 20 mg; D-Ile 50 mg;L-Phe 20 mg and 40 mg; L-Pro 50 mg and 100 mg; L-Ser 30 mg and 100 mg;L-Thr 30 mg; D-Trp 10 mg and 30 mg; L-Trp 8 mg; L-Tyr 10 mg; D-Tyr 10mg; L-Val 30 mg. TABLE 4 Comparison of D- and L-Amino Acids for FormingHydrogels L D Ala + − Arg {square root} −* Asn + − Asp − − Cys + Glu − −Gln + − Gly {square root} His {square root} − Hyp − Ile − + Leu − + Lys{square root} {square root} Met − + Nle + Phe + + Pro {square root}{square root} Ser + {square root} Thr + + Trp {square root} + Tyr + +Val + +{square root} = Gel formation when amino acid was dissolved in water;+ = Gel formation when amino acid was dissolved in 1 M NaHCO₃;− = No gel formation regardless of solvent for amino acid;*= HCl derivative of amino acid.

As shown in Table 4, some amino acids behave similarly, regardless ofwhether the L- or D-isomer is used. However, there is a significantnumber of amino acids that preferentially gel PVA as either the L- orthe D-isomer.

EXAMPLE 4

This Example describes studies to investigate the interactions betweenthe carboxylic acid and amino group of the amino acids and the hydroxylgroups of PVA.

As described in EXAMPLES 1-3, when solutions of PVA and amino acids aremixed, the system undergoes rapid gelation. This was unexpected becauseno chemical cross-linking or catalysis is required to produce the gelstate. The only functional moieties available on the polymer are thehydroxyl (OH) groups and the only available groups on amino acids suchas glycine and lysine are the α-amino (—NH₂), the carboxylate (COOH)and, in the case of lysine, the ε-amino (—NH₂) groups. The resultsobtained using succinic acid and ethylene diamine at different pHssupport the idea that hydrogen bonding occurs between the carbonyland/or amine groups of amino acids and the hydroxyl groups of PVA. Tofurther test this hypothesis, nuclear magnetic resonance (NMR) andinfrared spectroscopy (IR) was used to detect hydrogen bonding.

NMR spectroscopy is sensitive to influences of the chemical environmentsurrounding the atom of interest, either directly through the chemicalbond or through space. In polymer chemistry, NMR may be used to indicatechemical bond interactions, such as hydrogen bonding, apparent bychemical shifts of all carbon atoms whose substituents are involved inthe bond. ¹³C-NMR of PVA/Gly (1:1) hydrogels compared to PVA or glycinealone indicated the presence of hydrogen bonded COOH in PVA/Glyhydrogels, as shown in Table 5. Similar results were obtained withPVA/Arg (2:1) hydrogels, and PVA/Gly/Arg (formed using 480 mg PVA, 420mg Gly, and 225 mg Arg in 7 ml water containing 1% D₂O). Spectra wereobtained on a Bruker wm750, typically averaging >1024 scans. All aminoacid solutions and hydrogels contained 1% D₂O. The chemical shifts weremeasured in ppm. All measurements were made 24 hours after the PVA andamino acid solutions were combined. TABLE 5 NMR Spectra of Amino AcidSolutions and Hydrogels Carbonyl Carbon Gly Solution 172.8 PVA/GlyHydrogel 171.8 Arg Solution 183.5 PVA/Arg Hydrogel 182.9 Gly Solution172.8 Arg Solution 183.5 PVA/Gly/Arg Hydrogel 177.7 174.0

Similar to NMR spectroscopy, IR spectroscopy is sensitive to changes inthe chemical environment. In particular, O—H, C—O, and N—H stretchingmodes are significantly influenced by such interactions as hydrogenbonding. These changes are observed by broadening of the OH stretch inthe spectrum or by a shift of the signal as for the C═O stretch. Inaddition to these changes, appearance of overtone stretches fromhydrogen bonding of the amine and the hydroxyl groups were observed inthe PVA/aa spectra, as shown in Table 6. Therefore, the IR spectrum ofPVA/Gly (1:1), PVA/Arg (2:1), PVA/Arg/Gly (480 mg PVA, 225 mg Arg, 420mg Gly), and PVA/Lys (1:1) hydrogels indicated the presence of NH₃ ⁺hydrogen-bonding. TABLE 6 Summary of IR Results C═O C═O Sample OHStretch Overtones NH₃ ⁺ (free) (H-bonded PVA 3650-2900 − − − − Gly3350-2100 − − 1677 − Arg 3500-2750 − − 1669 1627 Lys 3600-2350 − 2145 −1585 PVA/Gly 3700-2350 {square root} 2130 1664 1618 PVA/Arg 3700-2350{square root} − 1676 1632 PVA/Arg/Gly 3650-2000 {square root} 2150 −1618 (br) PVA/Lys 3650-2000 {square root} 2126 − 1562 (br){square root} = Stretches observed in that region;− = No stretch observed;br = Broad stretch.

Therefore, characterization of PVA/aa hydrogels by NMR and IR supportsthe hypothesis that hydrogen bonding is responsible for the interactionbetween the carboxylic acid and amino group of the amino acids and thehydroxyl group of the PVA.

EXAMPLE 5

This Example describes the biocompatibility of representative hydrogelsof the invention.

A hydrogel comprising PVA and glycine was prepared by measuring 1.14 mLof 8% PVA into a vial, and injecting 1 ml of a 30% glycine solution intothe vial while mixing on a vortex. The hydrogel formed within less than30 seconds. The hydrogel was top-loaded into a syringe and was injectedinto the biceps femoris of C57B1/6 mice. The muscle was removed after 1,2, 4, and 8 weeks after implantation and the tissue prepared forhistological sectioning and staining. The tissue preparations showedthat the PVA/Gly hydrogel did not elicit any unfavorable responses suchas inflammation or foreign body encapsulation.

As another measure of biocompatibility, the amount of complement systemactivation of the PVA/amino acids (PVA/aa) hydrogels, in comparison toPVA and tissue culture polystyrene (TCPS) was examined. Results of thistype of in vitro study, provide a general guide to the performance ofthe materials in vivo.

PVA and PVA/aa hydrogel films were prepared by casting 500 microlitersof the final gel solution in a well of a 24-well plate. These wereallowed to dry for 4-5 days, hydrated with water and incubated with 9%NaCl solution. The samples were incubated with human serum from ahealthy donor for 90 min at 37° C. After addition of EDTA to a 10 mMfinal concentration, the serum was assayed for the presence of SC5b-9,which is the final membrane attack complex, resulting from both theclassical and alternative pathway. As a negative control, serum wasanalyzed immediately after thawing the serum to avoid activation. TCPSwas used as a positive control.

The results are provided in FIG. 3. The amount of complement activationobserved for TCPS and PVA was comparable to what has been previouslyfound (Black & Sefton (2000) Biomaterials 21(22):2287-94). No complementactivation was observed using PVA/aa hydrogels. This study providesadditional evidence that the PVA/aa hydrogels can be used asbiocompatible materials in biomedical devices.

EXAMPLE 6

This Example describes local gene delivery using a representativehydrogel of the invention.

Qualitative gene transfer from a representative hydrogel of theinvention comprising PVA and glycine (PVA/Gly) was observed in a murinemodel system of local gene delivery using the rAAV2-GFP vector. Theright biceps femoris muscle of 12 female C57B1/6 mice receivedapproximately 40 microliter of PVA/Gly prepared as described in EXAMPLE5, into which 1×10⁷ transducing units of an adenovirus associated viralvector encoding green fluorescent protein vector (rAAV2-GFP) wasinjected. The left biceps femoris muscle received approximately 40microliter of PVA/Gly alone. Mice were sacrificed at 1, 2, 4, and 8weeks. Three mice were sacrificed at each time point. Followingperfusion with 10 mL PBS, the biceps femoris muscles were dissected,rinsed with PBS, fixed in 4% paraformaldehyde for one hour, and embeddedin OCT for frozen sectioning. Eight micrometer thick longitudinalsections were made. The slides were placed in paraformaldehyde for 10minutes, rinsed with PBS, and cover-slipped with appropriate mountingmedia. Tissue sections were examined with fluorescence microscopy usinga Nikon E800 upright microscope at 10×/0.45 using a FITC filter cube.

The tissue sections showed that there was significant GFP expression wasat all time points in muscles injected with the vector compared tocontrol muscles injected with PVA/Gly alone. The amount of GFPexpression in muscles injected with the vector increased progressivelyover time.

EXAMPLE 7

This example describes the use of a representative hydrogel of theinvention as a barrier to extrusion of fluid from a site of injection.

Cardiovascular disease is the leading cause of death in the UnitedStates and worldwide. Injection of biologics (e.g., therapeutic genes orstem/progenitor cells) into damaged heart tissue holds promise fortreating myocardial ischemia by promoting angiogenesis and improving thefunctionality of damaged heart tissue. Minimally invasive surgeryrequires that biologics be delivered to a beating heart. However, backpressure, due to myocardial contraction, often extrudes a portion oftherapy into the pericardial space, which raises significant efficacyand safety concerns. Given its properties, it was hypothesized that ahydrogel of the present invention would create a self-sealing barrierthat would overcome the extrusion problem.

Initial studies involved testing the material as a “plug” just under thesurface of the heart muscle through which biologics could be injected.Toward this end, PVA/Gly hydrogel prepared using 400 microliters of 8%PVA and 350 microliters of 30% glycine was injected under the epicardiumusing a 25-gauge needle. Then 3 microliters of saline dyed withbromophenol blue was injected through the viscous PVA/Gly hydrogel intoheart muscle using a 30-gauge needle. In repeated attempts (both withhydrogels formed at about pH 6 and hydrogels formed at about pH 7), noneof the dye-saline solution was extruded from the myocardium into thepericardial space.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1-23. (canceled)
 24. A method for forming a hydrogel, comprisingcombining polymer molecules and bridging molecules to form a hydrogelwherein substantially all the polymer molecules are cross-linked byhydrogen bonds between polymer molecules and bridging molecules, whereineach bridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules.
 25. The method of claim 24, wherein the polymer molecules areselected from the group consisting of poly(vinyl alcohol), hydroxyethylacrylate, polyglyceryl acrylate, an acrylic co-polymer, andpolysaccharides.
 26. The method of claim 24, wherein the bridgingmolecules each comprise at least one of a carboxylic acid group or anamino group.
 27. The method of claim 24, wherein the bridging moleculesare selected from the group consisting of amino acids, succinic acid,and ethylene diamine.
 28. The method of claim 24, consisting essentiallyof combining polymer molecules and bridging molecules to form ahydrogel, wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules.
 29. The method of claim 28,wherein the polymer molecules are selected from the group consisting ofpoly(vinyl alcohol), hydroxyethyl acrylate, polyglyceryl acrylate, anacrylic co-polymer, and polysaccharides.
 30. The method of claim 28,wherein the bridging molecules each comprise at least one of acarboxylic acid group or an amino group.
 31. The method of claim 28,wherein the bridging molecules are selected from the group consisting ofamino acids, succinic acid, and ethylene diamine.
 32. The method ofclaim 24, consisting of combining polymer molecules and bridgingmolecules to form a hydrogel wherein substantially all the polymermolecules are cross-linked by hydrogen bonds between polymer moleculesand bridging molecules, wherein each bridging molecule is linked to atleast two polymer molecules, and wherein there are substantially nocovalent linkages between the polymer molecules.
 33. The method of claim32, wherein the polymer molecules are selected from the group consistingof poly(vinyl alcohol), hydroxyethyl acrylate, polyglyceryl acrylate, anacrylic co-polymer, and polysaccharides.
 34. The method of claim 32,wherein the bridging molecules each comprise at least one of acarboxylic acid group or an amino group.
 35. The method of claim 32,wherein the bridging molecules are selected from the group consisting ofamino acids, succinic acid, and ethylene diamine.
 36. A method forforming a hydrogel at a site of application, comprising combiningpolymer molecules and bridging molecules at a site of application toform a hydrogel wherein substantially all the polymer molecules arecross-linked by hydrogen bonds between polymer molecules and bridgingmolecules, wherein each bridging molecule is linked to at least twopolymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules.
 37. The method of claim 36,wherein the polymer molecules are selected from the group consisting ofpoly(vinyl alcohol), hydroxyethyl acrylate, polyglyceryl acrylate, anacrylic co-polymer, and polysaccharides.
 38. The method of claim 36,wherein the bridging molecules each comprise at least one of acarboxylic acid group or an amino group.
 39. The method of claim 36,wherein the bridging molecules are selected from the group consisting ofamino acids, succinic acid, and ethylene diamine.
 40. A method formaking a pharmaceutical composition, comprising combining biologicallyactive molecules with polymer molecules and bridging molecules to form ahydrogel comprising the biologically active molecules, whereinsubstantially all the polymer molecules are cross-linked by hydrogenbonds between polymer molecules and bridging molecules, wherein eachbridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules.
 41. The method of claim 40, wherein the polymer molecules areselected from the group consisting of poly(vinyl alcohol), hydroxyethylacrylate, polyglyceryl acrylate, an acrylic co-polymer, andpolysaccharides.
 42. The method of claim 40, wherein the bridgingmolecules comprise at least one of a carboxylic acid group or an aminogroup.
 43. The method of claim 40, wherein the bridging molecules areselected from the group consisting of amino acids, succinic acid, andethylene diamine.
 44. A method for administering biologically activemolecules to a subject, comprising administering to the subjectbiologically active molecules in a hydrogel, wherein the hydrogelcomprises polymer molecules and bridging molecules, whereinsubstantially all the polymer molecules are cross-linked by hydrogenbonds between polymer molecules and bridging molecules, wherein eachbridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules.
 45. The method of claim 44, wherein the polymer molecules areselected from the group consisting of poly(vinyl alcohol), hydroxyethylacrylate, polyglyceryl acrylate, an acrylic co-polymer, andpolysaccharides.
 46. The method of claim 44, wherein the bridgingmolecules each comprise at least one of a carboxylic acid group or anamino group.
 47. The method of claim 44, wherein the bridging moleculesare selected from the group consisting of amino acids, succinic acid,and ethylene diamine.
 48. A method for injecting biologically activemolecules, comprising the steps of: (a) applying a layer of hydrogel toa site of injection, wherein the hydrogel comprises polymer moleculesand bridging molecules, wherein substantially all the polymer moleculesare cross-linked by hydrogen bonds between polymer molecules andbridging molecules, wherein each bridging molecule is linked to at leasttwo polymer molecules, and wherein there are substantially no covalentlinkages between the polymer molecules; and (b) injecting biologicallyactive molecules through the hydrogel layer.
 49. The method of claim 48,wherein the polymer molecules are selected from the group consisting ofpoly(vinyl alcohol), hydroxyethyl acrylate, polyglyceryl acrylate, anacrylic co-polymer, and polysaccharides.
 50. The method of claim 48,wherein the bridging molecules each comprise at least one of acarboxylic acid group or an amino group.
 51. The method of claim 48,wherein the bridging molecules are selected from the group consisting ofamino acids, succinic acid, and ethylene diamine.
 52. A kit, comprisingpolymer molecules, bridging molecules, and instructions for forming ahydrogel, wherein the instructions provide protocols for combining thepolymer molecules and bridging molecules to form a hydrogel whereinsubstantially all the polymer molecules are cross-linked by hydrogenbonds between polymer molecules and bridging molecules, wherein eachbridging molecule is linked to at least two polymer molecules, andwherein there are substantially no covalent linkages between the polymermolecules.
 53. The method of claim 52, wherein the polymer molecules areselected from the group consisting of poly(vinyl alcohol), hydroxyethylacrylate, polyglyceryl acrylate, an acrylic co-polymer, andpolysaccharides.
 54. The method of claim 52, wherein the bridgingmolecules each comprise at least one of a carboxylic acid group or anamino group.
 55. The method of claim 52, wherein the bridging moleculesare selected from the group consisting of amino acids, succinic acid,and ethylene diamine.